Unraveling the role of Xist in X chromosome inactivation: insights from rabbit model and deletion analysis of exons and repeat A

X chromosome inactivation (XCI) is a process that equalizes the expression of X-linked genes between males and females. It relies on Xist, continuously expressed in somatic cells during XCI maintenance. However, how Xist impacts XCI maintenance and its functional motifs remain unclear. In this study, we conducted a comprehensive analysis of Xist, using rabbits as an ideal non-primate model. Homozygous knockout of exon 1, exon 6, and repeat A in female rabbits resulted in embryonic lethality. However, X∆ReAX females, with intact X chromosome expressing Xist, showed no abnormalities. Interestingly, there were no significant differences between females with homozygous knockout of exons 2–5 and wild-type rabbits, suggesting that exons 2, 3, 4, and 5 are less important for XCI. These findings provide evolutionary insights into Xist function. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-024-05151-0.


Introduction
X chromosome inactivation (XCI) is a dosage compensation mechanism that evolved in marsupial and placental mammals to equalize the expression of X-linked genes between females (XX) and males (XY) [1][2][3][4][5].The initiation of XCI is genetically controlled by a master regulatory locus called the X-inactivation center (Xic) [6][7][8][9][10].In mice and humans, dosage compensation is mediated by a long noncoding RNA (lncRNA) called Xist.Xist is up-regulated from one of the two X chromosomes and its RNA accumulates over the inactive X chromosome (Xi) to trigger gene silencing [7,9,11].Once established, XCI is stably inherited upon successive cell divisions in female somatic cells.Extensive studies have confirmed that Xist is both necessary and sufficient for XCI [12][13][14].
A significant amount of our current understanding of X chromosome inactivation (XCI) mechanisms comes from studies conducted on mice and in vitro female stem cells [5,45,46].The gene Xist, which is involved in XCI, exhibits species-specific differences in its regulation and function.In humans, XIST is expressed on both X chromosomes, which undergo random XCI during cell differentiation [47,48].In contrast, mice initially exhibit paternal Xist expression during X chromosome inactivation, followed by random X chromosome inactivation during subsequent stages.Therefore, there are notable differences between the mechanisms of X inactivation in mice and humans [48].Consequently, it is crucial to establish suitable animal models for investigating the functions of XIST and studying the mechanisms of XCI.
According to a study published in Nature by Okamoto et al., rabbits display a similar XCI mechanism to humans during early embryogenesis [48].In both species, X chromosome inactivation occurs randomly during both stages of XIST expression.However, in contrast, the house mouse exhibits paternal Xist expression during the initial stage of X chromosome inactivation, followed by random X chromosome inactivation during the subsequent stage (Fig. 1D).These findings emphasize the variations in the XCI mechanism that regulate Xist expression in mice and humans.Therefore, the rabbit serves as an ideal animal model for studying XCI.
In this study, we performed a thorough phylogenetic analysis that unveiled a strong connection between rabbits and primates.Moreover, our analysis demonstrated that human XIST shares a greater sequence similarity with rabbits than with mice.This suggests that rabbits would serve as an excellent animal model for investigating the XCI mechanism.Additionally, we employed CRISPR/Cas9 to delete exon 1-6 and repeat A of the rabbit Xist RNA transcript, enabling us to determine its functionality.In conclusion, these findings enhance our comprehension of the functional mechanisms involved in Xist-induced XCI at the animal level.

Animals care and use
The Institutional Animal Care and Use Committee of Jilin University approved all animal experiments.New Zealand White rabbits were obtained from the Laboratory Animal Centre of Jilin University (Changchun, China).All animal experiments were conducted by the guidelines for animal experiments of the Laboratory Animal Center of Jilin University.

Plasmid design and construction
Eleven pairs of sgRNAs were designed to knock out Xist different regions according to the previous description [49], which were cloned into the BbsI-linearized pUC57-T7-gRNA vector.Then, sgRNAs were amplified using PCR with T7 primers (T7-Fwd: 5′-GAA ATT AAT ACG ACT CAC TAT A-3' and T7-Rev: 5′-AAA AAA AGC ACC GAC TCG GTG CCA C-3') and in vitro transcribe using the MAXIscript T7 kit (Invitrogen) and purified with a miRNeasy mini kit (QIAGEN) according to the manufacturer's instructions.To produce SpCas9 mRNA, the PCS2 + Cas9 (Plasmid #122,948) plasmid was linearized with NotI restriction digestion and used as a template to in vitro transcribe mRNAs using mMESSAGE mMA-CHINE SP6 Transcription Kit (Invitrogen) and then Cas9 mRNAs were purified with a miRNeasy mini kit (QIA-GEN) according to the manufacturer's instructions.All sgRNA sequences are listed in Supplementary Table 1.

Microinjection of rabbit zygotes
The protocol for microinjecting sgRNA/Cas9 mRNA into pronuclear stage embryos is detailed in our previously published study [50].Briefly, a mixture of Cas9 mRNA (200 ng/ul) and sgRNA (50 ng/ul) was co-injected into the cytoplasm of pronuclear stage zygotes.Finally, 40-50 injected zygotes were transferred into the oviduct of recipient rabbits.

Single-embryo and rabbit genotyping by PCR
The zygotes injected with sgRNA/Cas9 mRNA were cultured for 4 days and subsequently collected for genotyping analysis.Embryos were incubated in lysis buffer at 50 °C for 20 min and then at 90 °C for 5 min in a PCR machine.Genomic DNA was extracted from newborn rabbits for PCR genotyping, followed by Sanger sequencing and T-A Fig. 1 Comparison of Xist among different species.A Evolutionary relationships of the XIST/Xist gene among Primates, Lagomorpha, Artiodactyla, and Rodentia.Mammalian phylogeny was estimated using maximum likelihood from 18 nucleotide sequences.Clades discussed in the text are labeled.Bootstrap support values ≥ 88% are indicated at nodes.The scale bar indicates evolutionary distance.B Xist sequences were aligned to humans using the NCBI BLAST server.C Dot plot analysis of Xist/XIST cDNA sequences in mouse, rabbit, and human using the EMBOSS dot-matcher program.D Hypothesis explaining differences in Xist/XIST regulation and XCI initiation observed in mouse, rabbit, and human embryos, based on previous work [48].E Schematic representation of the sequence homology of Xist/XIST exons across mice, humans, and rabbits.The percent identity mentioned in the text is indicated.F Schematic diagram illustrating the establishment of a cloned rabbit model using standard microinjection procedures.G Schematic representation of the Xist knock-out created in rabbits using CRISPR/Cas9; the different exons are highlighted in colored boxes, with repeat A highlighted in the red box.Deleted exons are represented as white boxes ◂ cloning.Please refer to Supplementary Table 1 for a list of all primers.

RT-PCR and quantitative real-time PCR analysis
Tissue RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's instructions, and cDNA synthesis was performed on extracted RNA using FastKing cDNA First Strand Synthesis Kit (TIANGEN, KR116).Embryo RNA was extracted using the MicroElute® Total RNA Kit (Omega, R6831) according to the manufacturer's instructions, and cDNA synthesis was performed on extracted RNA using PrimeScript™ RT Master Mix (Takara, RR036A).A QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) was utilized for conducting quantitative real-time PCR experiments.For each gene, three biological replicates and three technical replicates (3 × 3) were carried out.The GAPDH gene was employed as an internal control to standardize the expression data.The gene-specific primers for RT-PCR and qRT-PCR can be found in Supplementary Table 2 and 3, respectively.

Hematoxylin and eosin (H&E) staining
The hematoxylin and eosin (H&E) staining was performed according to our published protocols [51].Briefly, the tissues from WT and mutant rabbits were fixed in 4% paraformaldehyde for 48 h, embedded in paraffin wax, and then sectioned for slides.The slides were stained with hematoxylin and eosin, and were viewed under a Nikon TS100 microscope.

Statistical analysis of weight and survival
To analyze survival, we conducted regular daily monitoring of the rabbits.The survival data are from 6 KO rabbits and 6 control rabbits.Body weight was recorded weekly.All data are expressed as mean ± SEM from at least three determinations in all experiments.The data were analyzed by Student's unpaired t-test using GraphPad Prism software.p < 0.05 indicated statistical significance ( * p < 0.05, * * p < 0.01, * * * p < 0.001).

Phylogenetic tree construction
To conduct the phylogenetic analysis of lncRNA Xist, we downloaded all Xist sequences of various species from the NCBI database.Using MEGA, we constructed maximum likelihood phylogenetic trees, with 1000 bootstrap replicates [52][53][54].The tree is drawn to scale, and the branch lengths (next to the branches) are in the same units as the evolutionary distances used to infer the phylogenetic tree [52].

Dot plots
To access sequence similarity, dot plots were generated using EMBOSS dot-matcher [55].To enhance visualization clarity, two different thresholds were utilized to generate distinct dot plots.In this analysis, all positions of the first input sequence were systematically compared with all positions of the second input sequence using a specified substitution matrix.The resulting dot plots were generated as a rectangular grid, with the two sequences serving as the axes.Each dot in the plot represents a position where a similarity was identified between the corresponding positions of the two sequences.

RNA secondary structure
The secondary structure graph is created using the Vien-naRNA Package from RNA secondary structure predictions [57].The red circles (bases) indicate a confidence level of 90% or higher, based on minimum free energy (MFE) and partition function.

Rabbits are the ideal non-primate animal model for studying Xist in vivo
To investigate the functional role of Xist, we performed a phylogenetic analysis using Molecular Evolutionary Genetics Analysis software (MEGA11).We employed the maximum composite likelihood method with 1000 bootstrap replicates [58].Reference sequences of the Xist gene were obtained from the NCBI database [59].The phylogenetic tree analysis of the XIST/Xist gene showed a close relationship between rabbit species and humans, while excluding non-human primates (Fig. 1A).Additionally, the three species, namely pigs, cows, and sheep, were found to belong to the same sister branch in the taxonomic status of the evolutionary tree, indicating a close evolutionary relationship.To validate our evolutionary classification, we used various methods to analyze the phylogenetic relationships based on different theoretical models between species (Fig. S1A and B).These results were consistent with the findings observed in Fig. 1A.The results revealed that rabbits and primates are part of the same main branch of the phylogenetic tree, indicating a closer relationship of the XIST/Xist gene between rabbits and primates.
Additionally, we compared the Xist DNA sequences of different non-primate species.Our findings show that rabbits have the highest overall score and sequence coverage, with a total score of 17,048 and a coverage of 94%.In contrast, the house mouse, which is a commonly used animal model, scored only 4443 with a coverage of 40%, indicating a lower DNA homology to human Xist.This suggests that rabbit Xist has a higher similarity to humans (Fig. 1B).Furthermore, we also observed a higher homology of pig Xist to humans.To further understand the sequence conservation among humans, rabbits, and pigs, we conducted multiple sequence alignments (Fig. S1C).In the alignment results, we noticed a significant region of low similarity in the Xist sequence of pigs, spanning from 4700 to 15,600.Although some local regions show higher similarity to humans, the overall genomic structural homogeneity is lost in pig Xist.Additionally, the genetic distance matrix confirms that the Xist sequence of rabbits is more closely related to humans (Fig. S1D).This finding aligns with the evolutionary relationship between species [60][61][62].
To further evaluate the similarity of Xist sequences among the house mouse, rabbit, and human, we conducted a dot-plot analysis [55].We consistently obtained results across various window sizes and thresholds, confirming that rabbits exhibit a higher sequence similarity to humans than mice (Fig. 1C).
In addition, rabbits exhibit a similar XCI mechanism to humans during early embryogenesis [48].Both humans and rabbits undergo random X chromosome inactivation during both stages of XIST expression.On the other hand, mice display paternal Xist expression during the initial stage of X chromosome inactivation, followed by random X chromosome inactivation during the subsequent stage (Fig. 1D).
Then, we conducted a comparative analysis to assess the homology of exons among mice, rabbits, and humans XIST/ Xist (Fig. 1F and Fig. S3A).The results showed that human XIST exon 1 had a higher homology of 77.07%with rabbit Xist exon 1 compared to the 67.28% homology observed with mouse Xist exon 1.Interestingly, we did not find any homologous sequence of human XIST exon 2 in either mice or rabbits.On the other hand, human XIST exon 3 showed a homology of 76.80% with rabbit Xist exon 2, but no homologous sequence was detected in mice.Furthermore, human XIST exon 4 had a higher homology of 91.46% with rabbit Xist exon 3 compared to the 79.80% homology with mouse Xist exon 4. Additionally, human XIST exon 5 demonstrated homologies of 74.59% and 74.42% with mouse Xist exon 6 and rabbit Xist exon 5, respectively.Similarly, human XIST exon 6 showed homologies of 70.85% and 70.13% with mouse Xist exon 7 and rabbit Xist exon 6, respectively.
Besides, we conducted a comparison to determine the similarity of other repeat sequences on the Xist loci in human, mouse, and rabbit (Fig. S2A).Initially, we assessed the homology between different repeat sequences and found that the homology between rabbit repeat F and human repeat F was 85.71%.However, no corresponding homologous sequence of human repeat F was found in mice.Similarly, the homology between rabbit repeat C and human repeat C was 76.47%, but no corresponding homologous sequence was identified in mice.The homology between rabbit repeat D and human repeat D was 72.77%, while mouse repeat D exhibited 79.12% homology to its human counterpart.
Rabbit repeat E showed a 71.23% homology to the human sequence, whereas no corresponding homologous sequence was found in mice (Fig. S2B).Furthermore, we compared the motif similarity of the different repeat sequences and observed that the rabbit repeats were more similar in length to human repeats (Fig. S2C).Notably, repeat B consisted of repeat units "enriched in cytosine bases," and repeat E exhibited greater similarity between rabbit and human sequences.In contrast, the mouse sequence exclusively consisted of repeat units "enriched in thymine bases."These findings position rabbits as an ideal non-primate animal model for studying Xist in vivo.
In summary, these findings highlight the significance of rabbits as an invaluable model for understanding the functions of Xist.To generate cloned animals, we co-injected Cas9 mRNA and sgRNA into one-cell stage embryos and transferred them into surrogate mother rabbits (Fig. 1E and Fig. S3B).We utilized CRISPR/Cas9 technology to disrupt the exon sequences of Xist in rabbits [63,64].Specifically, we targeted exons 1-6 and repeat A to investigate the functional role of Xist (Fig. 1G).

Deletion of exon 1 in female rabbits does not survive
Sequence homology analysis was conducted on Xist exon 1 in mice, humans, and rabbits using the NCBI BLAST service.The coverage rate of rabbit Xist exon 1 was determined to be 95% when compared to the human XIST sequence, which was significantly higher than the 41% observed in mice.Additionally, the percentage identity between rabbit Xist exon 1 and human XIST exon 1 was found to be 77.07%,while the percentage identity between mice and humans was 67.28% (Fig. 2A).Dot-plot analysis confirmed that rabbit Xist exon 1 showed greater homology to humans compared to mice (Fig. 2B).
To produce knock-out rabbits using CRISPR/Cas9 technology, we designed four single-guide RNAs (sgRNAs) that targeted exon 1 of Xist.Genotyping was conducted by PCR  1. D Founder rabbits from the F0 generation are identified through agarose gel electrophoresis.E The gross appearance of rabbits from the F0 generation at day 7 reveals that Xist exon 1 knock-out rabbits exhibit developmental delay.F, left Schematic for generating heterozygous Xist exon 1 deletants.(Right) Genotype data for F0; the number of pups for each genotype is listed.G The body weight of male X △Ex1 Y F0 rabbit and male littermate controls (n = 4).Error bars indicate mean ± SEM.H The survival curve for X △Ex1 Y and male littermate controls.I H&E staining for cardiac muscle from X △Ex1 Y and male control animal.Scale bars, 50um using four sets of primers specific to certain genes, including the Sry gene, which is only present in males.Sanger sequencing was then performed to confirm the genotyping results (Fig. 2C and D).Among the genotyping results of the founder animals (Fig. 2F), we did not identify any homozygous knockout female rabbits.However, we were successful in obtaining a hemizygous knockout male rabbit, named #5 (Fig. 2E).Unfortunately, this male rabbit showed developmental delays (Fig. 2G) and eventually died at 25 weeks (Fig. 2H).Examination of the #5 rabbit's myocardium revealed a loose arrangement of cardiac fibers (Fig. 2I).
Given the consistent challenges encountered in obtaining homozygous knockout female individuals, our hypothesis was that the X ∆Ex1 X ∆Ex1 homozygous mutant females could be generated but would die early in embryogenesis.
To investigate this possibility, we conducted an embryoniclevel investigation by employing a fibroblast injection method to introduce sgRNA and Cas9 RNA into zygotes.After a week-long incubation period, genotyping was performed using PCR and Sanger sequencing.Remarkably, the results revealed successful large-scale deletions at the embryonic level (Fig. S3C and D).The results showed that the X ∆Ex1 X ∆Ex1 homozygous mutant females can be generated but perish early in embryogenesis.

Deletion of Xist repeat A in rabbits results in embryonic lethality.
To investigate the impact of Xist repeat A on individual development, we conducted a comparative analysis of sequence homology in mice, humans, and rabbits.Interestingly, our findings showed that the rabbit and human repeat A sequences had a higher level of homology (78.44%), while the homology between mouse and human sequences was 67.48% (Fig. 3A).This was further supported by the results of the dot-plot analysis, which confirmed that the rabbit repeat A sequence bore a closer resemblance to the human sequence (Fig. S4A and Fig. 3B).Moreover, a more similar pattern of repeat motifs was observed in rabbits and humans (Fig. 3C and Fig. S4B).Additionally, the major stem-loop structures in both rabbit and human repeat A sequences were consistent, whereas the mouse sequence showed discrepancies (Fig. 3D).Specifically, the RNA hairpin in rabbit and human sequences consisted of a 12-nucleotide AUCG tetraloop, while the mouse sequence had an AWCG tetraloop.These results clearly demonstrate that the rabbit Xist repeat A sequence is more similar to the human sequence.Subsequently, two pairs of single guide RNAs (sgRNAs) were designed to target and delete the Xist repeat A. The genotyping was determined by performing PCR, and the results were further confirmed through Sanger sequencing (Fig. 3E).
To determine the viability of homozygote knockout Xist repeat A (X ∆ReA X ∆ReA ) females, we attempted to generate offspring through hybridization (Fig. 3F and Fig. S4C-G).Despite multiple attempts, we were unsuccessful in producing X ∆ReA X ∆ReA female rabbits (Fig. 3F and Fig. S4D).
To investigate the timing of development cessation in X ∆ReA X ∆ReA females, we partially tracked early embryonic development.Initially, we examined early embryos at rabbit embryonic day 12 (E12) for characterization.The results indicated the absence of homozygote individuals.Interestingly, all heterozygous individuals exhibited skewed X chromosome inactivation, as evidenced by the transcription of Xist RNAs from intact X chromosomes (Fig. S5A-C).Subsequently, we analyzed embryos from the E9.5 period and obtained similar outcomes; no homozygote individuals were present, and all heterozygous individuals showed skewed X chromosome inactivation (Fig. S5D and E).
Furthermore, we observed no significant differences in body weight (Fig. 3G), survival rates (Fig. 3H), and X-linked gene expression levels (Fig. 3I) between X ∆ReA X females and WT rabbits.The normal development of the heart, liver, spleen, lungs, and kidneys in X ∆ReA X females was comparable to that of WT rabbits (Fig. S4H).
RT-PCR results confirmed the previous observation of skewed X chromosome inactivation in all samples (Fig. 3J).These findings suggest that Xist repeat A is transcribed from the complete X chromosome (Fig. 3K).Additionally, the average number of offspring in the X ∆ReA X female and X ∆ReA Y male cross-group (5.071 offspring) was lower than that in the WT group (7.118 offspring), further supporting the conclusion that embryos lacking Xist repeat A function do not survive (Fig. 3L).

Deletions of Xist exon 2 in rabbits are viable and develop normally
Analysis of sequence homology revealed that there is no homologous sequence of human XIST exon 3 in mice.However, there is a higher homology (76.80%) between rabbit Xist exon 2 and human XIST exon 3 (Fig. 4A), which was also confirmed by dot plot analysis (Fig. S6A).Therefore, deletions of Xist exon 2 in rabbits were used as a model to mimic the function of XIST exon 3 in humans.
Seven founder (F0) pups were identified using Sanger sequencing.We obtained a chimeric male with exon 2 deletion (Fig. 4B and C).By further backcrossing, we successfully produced female homozygous knockout rabbits (Ex2 −/− ) in the F2 generation (Fig. 4D and Fig. S6B).RT-PCR results confirmed the absence of exon 2 sequence in the expressed Xist RNA of the rabbits (Fig. 4F).It is noteworthy that there were no significant differences in terms of body weight (Fig. 4G), survival rates (Fig. 4H), X-linked gene expression (Fig. 4I), and reproductive efficiency (Fig. 4E) between the Ex2 −/− and WT rabbits.These findings indicate that the Ex2 −/− rabbits exhibit normal growth and development.

Deletions of Xist exon 3 in rabbits are viable and develop normally
The results of the sequence homology analysis revealed that there was a 79.80% sequence homology between rabbit Xist exon 3 and human XIST exon 4, with a coverage of 94%.This was significantly higher compared to the 76.80% sequence homology and 89% coverage observed between human XIST exon 4 and mouse Xist exon 4 (Fig. 5A and Fig. S6C).These findings were further confirmed by the dot plot analysis (Fig. 5B).
By using Sanger sequencing, we were able to identify the founder (F0) pups, and obtain a male chimeric rabbit with exon 3 knockout and a single knockout female rabbit (Fig. 5C and D).Subsequent breeding allowed us to successfully obtain female homozygous knockout rabbits (Ex3 −/− ) in the F1 generation (Fig. 5E and Fig. S5D).The RT-PCR results demonstrated the absence of exon 3 sequence in the expressed Xist RNA of the rabbits (Fig. 5G).Importantly, there were no significant differences observed in terms of body weight (Fig. 5H), survival rates (Fig. 5I), X-linked gene expression (Fig. 5J) and reproductive efficiency (Fig. 5F) between the Ex3 −/− and WT rabbits.These findings indicate normal growth and development of the Ex3 −/− rabbits.

Deletions of Xist exon 4 in rabbits are viable and develop normally
Rabbit Xist exon 4 is the only exon that does not have sequence homology with its human counterpart.To investigate the functionality of rabbit Xist exon 4, we employed a targeted approach using a pair of sgRNAs to specifically disrupt this exon.The F0 generation rabbits were identified using Sanger sequencing, and we obtained a male chimeric individual with a complete knockout of exon 4 and a single knockout female individual (Fig. S7A and B).By subsequent breeding, we successfully obtained female homozygous knockout rabbits (Ex4 −/− ) in the F1 generation (Fig. S7C and Fig. S6E).RT-PCR results demonstrated the absence of exon 4 sequence in the expressed Xist RNA of the rabbits (Fig. S7E).Importantly, we found no significant differences in terms of body weight (Fig. S7F), survival rates (Fig. S7G), X-linked gene expression (Fig. S7H), and reproductive efficiency (Fig. S7D) between the Ex4 −/− and WT rabbits.These findings indicate normal growth and development of the Ex4 −/− rabbits.

Deletions of Xist exon 5 in rabbits are viable and develop normally
Sequence homology analysis revealed that rabbit Xist exon 5 had a 74.42% sequence homology with human XIST exon 5, with 100% coverage.This was significantly higher than the 74.59% and 78% sequence homology observed between human XIST exon 5 and mouse Xist exon 6 (Fig. 6A and Fig. S6F).Dot plot analysis confirmed these findings (Fig. 6B).
Sanger sequencing identified the founder (F0) pups, and homozygous exon 5 knockout male and female rabbits were obtained (Fig. 6C and D).Breeding subsequently led to the generation of F1 rabbits (Fig. 6E).RT-PCR results demonstrated the absence of exon 5 sequence in the expressed Xist RNA of the rabbits (Fig. 6G).Importantly, no significant differences were observed in terms of body weight (Fig. 6H), survival rates (Fig. 6I), X-linked gene expression (Fig. 6J), and reproductive efficiency (Fig. 6F) between the Ex5 −/− and WT rabbits.These findings indicate normal growth and development of the Ex5 −/− rabbits.

Deletion of Xist exon 6 in rabbits results in embryonic lethality
The results of the sequence homology analysis showed that there was a 70.13% sequence homology between rabbit Xist exon 6 and human XIST exon 6, with a coverage of 95%.This was significantly higher compared to the 70.85% sequence homology and 39% coverage observed between human exon 6 and mouse Xist exon 7 (Fig. 7A).These findings were also confirmed by dot plot analysis (Fig. 7B).Thus, the deletions of Xist exon 6 in rabbits were used to mimic the function of XIST exon 6 in humans.
To disrupt the function of Xist exon 6 in rabbits, we designed two pairs of sgRNAs targeting Xist exon 6 (Fig. 7C).The F0 generation rabbits were subsequently confirmed through PCR and Sanger sequencing.Surprisingly, no Xist exon 6 knockout animals were obtained from pregnant females (Fig. 7D and E), indicating that the absence of exon 6 resulted in non-viability.Additionally, statistical analysis of offspring production revealed a reduced number of offspring from pregnant females compared to wild-type (WT) rabbits (Fig. 7F).
To further characterize the lethal stage of early embryonic development, we injected Cas9 mRNA and sgRNA Fig. 3 Xist repeat A homozygous knockout rabbit does not survive.A The BLAST result compares the Xist/XIST repeat A sequences in mouse, human, and rabbit.B Dot plot analysis illustrates the sequence similarities between mouse, rabbit, and human Xist/XIST repeat A. C The top motifs identified by MEME in Xist/XIST repeat A are compared, with the number of sites and E-value displayed below each motif logo.D Secondary structure analysis of Xist/XIST repeat A is presented for human, rabbit, and mouse.The total numbers of stem loops in repeat A are listed.E Target loci and Sanger sequencing results provide evidence of Xist repeat A knock-out in F0 rabbit.All sgRNA sequences are listed in Supplementary Table 1.F A schematic depicts the process of generating homozygous Xist repeat A deletants.Genotype data for F3; the number of pups for each genotype is listed.G Body weight of X △ReA X and WT rabbits.Error bars indicate mean ± SEM.H Survival curve for X △ReA X and WT rabbits.I Expression of X-linked genes in X △ReA X and WT rabbits.Error bars indicate mean ± SEM.J Gene expression analysis by RT-PCR.K A model for Xist-mediated transcriptional silencing across the X chromosome in X △ReA X rabbits.L Newborn data from cross-breeding between X △ReA X / X △ReA Y compared to WT control (box and whiskers plot, min.to max., all points shown) ◂ Fig. 4 Viability of Xist exon 2 knockout rabbits.A Sequence alignment comparing human XIST exon 3 and rabbit Xist exon 2, with identical bases highlighted on a dark background.B Sanger sequencing results and target loci confirm Xist exon 2 knock-outs in F0 rabbits.All sgRNA sequences are listed in Supplementary Table  into fertilized zygotes.Surprisingly, we were able to successfully knockout rabbit Xist exon 6 in early-stage embryos (Fig. 7G and Fig. S6G).However, our results revealed that the cloned embryos had a significantly lower blastocyst rate (13.3 ± 0.9%) compared to the control group (74.7 ± 5.4%).These findings provide conclusive evidence that the deletion of Xist exon 6 leads to embryonic lethality in rabbits, hindering proper development.

Discussion
Xist is continuously expressed in female somatic cells to maintain X chromosome inactivation (XCI) [65].Previous studies on Xist's functional domains have primarily used mouse models and cellular-level experiments.However, it is important to note that mice display paternal Xist expression during the initial stage of XCI, followed by random X chromosome inactivation during the subsequent stage,    Representative images of agarose gel electrophoresis display the PCR products from the F0 generation.F The newborn data from the F0 generation is compared to the WT control using a box and whiskers plot, showing the minimum to maximum values and t-test results (P = 0.0001).G The target loci and Sanger sequencing results confirm the knock-out of Xist exon 6 in rabbit embryos which differs significantly from the random XCI mechanism observed in humans [66,67].Therefore, the consistency of cellular-level results in vivo remains uncertain.Initially, we chose to focus on the homozygous deletion of different exons due to the high degree of homology among different species.However, the exact functional mechanism was unclear, especially regarding rabbit Xist exon 2, which corresponds to human XIST exon 3.In mice, there is no homologous sequence for human XIST exon 3. Therefore, we embarked on a systematic characterization of these exons.Subsequently, numerous studies suggest that repeat A is crucial for gene silencing.Deletion of repeat A has been found to result in X chromosome silencing failure [24,25,42,68,69].However, the implications of this finding in vivo are unknown.In our study, we deleted repeat A in rabbits and found that individuals homozygous for the deletion exhibited embryonic lethality and were unable to develop.Interestingly, individuals heterozygous for the repeat A deletion transcribed the Xist gene from the intact X chromosome, indicating skewed X chromosome inactivation.This suggests that the choice of which X chromosome to inactivate is consistent across all early embryonic stem cells, either Xp or Xm, rather than a combination of the two.There were no phenotypic differences compared to individuals with the wild-type genotype.When we analyzed the reproductive quantities in the offspring, we found a lower number compared to the control group, suggesting that X ∆ReA X ∆ReA or X ∆ReA X (Xist is transcribed by X ∆ReA ) may lead to embryonic lethality.These findings indicate that repeat A plays a crucial role in establishing X chromosome inactivation and female development.
Deletion of the 5' conserved region of Xist, which includes exon 1, in mice revealed that female mice lacking Xist RNA were able to develop and survive until birth [70].However, there was a lower frequency of female births and they had a smaller size at birth, although most organ development was normal.In our study, when exon 1 was deleted in rabbits, it led to delayed development and premature death in males.Histological analysis further revealed impaired heart development.Deletion of Xist exon 1 had an impact on male development and led to decreased birth rates.Moreover, females were found to be incapable of surviving, indicating the crucial role of exon 1 in the process of X-chromosome inactivation in females.Specifically, the deletion of exon 1 hindered both the development and survival of females.
The functionality of regions other than Xist repeat A is currently poorly understood.According to a few reports, repeat B and C play a critical role in recruiting epigenetic modifier proteins to maintain the epigenetic state of XCI [35], while repeat E is essential for Xist localization and gene silencing [41].However, the functions of other regions are still unknown.In a previous study, it was suggested that XIST exon 5 is crucial for maintaining XCI status in human K562 cells, while exons 2, 3, and 4 seem to be dispensable [44].However, the implications of these findings in vivo remain unknown.The results of the study indicate that rabbits lacking these exons can be born and survive normally, showing no significant differences in body weight, survival rate, or X-linked gene expression compared to WT individuals.This suggests that these exons may not be necessary for normal functioning in living organisms.Moreover, the previously emphasized importance of exon 5 in maintaining XCI status at the cellular level seems to have little significance in vivo.Additionally, the functional role of Xist exon 6 remains unknown.Our study found that the deletion of exon 6 in living organisms led to a lower rate of embryo blastocysts and the absence of offspring lacking exon 6.These findings demonstrate the vital and essential role of Xist exon 6 in embryonic development and individual survival.In conclusion, our study comprehensively elucidated the functional roles of Xist exons and repeat A in vivo, enhancing our understanding of the functional landscape of different regions within Xist and offering new insights into the functional mechanisms of Xist in X chromosome inactivation.included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this licence, visit http:// creat iveco mmons.org/ licen ses/ by/4.0/.

Fig. 2
Fig. 2 Male rabbit with Xist exon 1 deletion exhibit a loose arrangement of the myocardial fibers.A The BLAST results of Xist/XIST exon 1 are compared between mouse, human, and rabbit.B Dot plot analysis of the Xist/XIST exon 1 sequence in mouse, rabbit, and human.C Target loci and Sanger sequencing results demonstrate the knock-out of Xist exon 1 in F0 male rabbits.All sgRNA sequences are listed in Supplementary Table1.D Founder rabbits from the F0 generation are identified through agarose gel electrophoresis.E The gross appearance of rabbits from the F0 generation at day 7 reveals

1
Fig.4 Viability of Xist exon 2 knockout rabbits.A Sequence alignment comparing human XIST exon 3 and rabbit Xist exon 2, with identical bases highlighted on a dark background.B Sanger sequencing results and target loci confirm Xist exon 2 knock-outs in F0 rabbits.All sgRNA sequences are listed in Supplementary Table1.C Agarose gel electrophoresis result of PCR product in the F0 generation.(D) Schematic illustrating the generation of the F1 and F2 generations.E, up Schematic diagram outlining the breeding strategy employed to generate the F3 generation.Genotype data for F3, along with the number of pups for each genotype, is provided.(Down) Aga-

Fig. 5
Fig.4 Viability of Xist exon 2 knockout rabbits.A Sequence alignment comparing human XIST exon 3 and rabbit Xist exon 2, with identical bases highlighted on a dark background.B Sanger sequencing results and target loci confirm Xist exon 2 knock-outs in F0 rabbits.All sgRNA sequences are listed in Supplementary Table1.C Agarose gel electrophoresis result of PCR product in the F0 generation.(D) Schematic illustrating the generation of the F1 and F2 generations.E, up Schematic diagram outlining the breeding strategy employed to generate the F3 generation.Genotype data for F3, along with the number of pups for each genotype, is provided.(Down) Aga-

Fig. 6 Fig. 7
Fig.6 Viability of Xist exon 5 knockout rabbits.A BLAST analysis comparing human XIST exon 5, rabbit Xist exon 5, and mouse Xist exon 6. B Dot plot analysis of mouse, rabbit, and human sequences.C Sanger sequencing results and target loci confirm Xist exon 5 knockouts in F0 rabbits.All sgRNA sequences are listed in Supplementary Table1.D Agarose gel electrophoresis result of PCR product in the F0 generation.E Schematic representation of the process for generating the F1 generation.F, left Genotype data for F2 rabbits; the